N-acetylglutamate (NAG) is an essential allosteric activator of carbamyl phosphate synthetase I (CPSI), the first enzyme of the urea cycle. However, the study of this system has been hampered by lack of information on the mammalian NAGS gene. Our recent cloning of the mouse and human N-acetylglutamate synthase (NAGS) genes, characterization of their structures, purification of the respective recombinant proteins and development of accurate methods to measure NAG and NAGS activity now permit investigations on the role of this enzyme in health and disease. The specific aims of this study are: 1. To determine domains critical to catalytic function of recombinant human NAGS using random mutagenesis. Chemical and PCR mutagenesis will be used to alter the human NAGS gene, followed by complementation in NAGS deficient E. coil (NK5992) and yeast arg2 strains and biochemical studies of the recombinant mutant enzymes. 2. To characterize the mouse and human NAGS mitochondrial targeting signal (MTS). Affinity purification of native mouse and human liver NAGS will be used to determine the amino-terminus. Subsequently, N-terminal deletion and fusion NAGS constructs will be used for complementation in yeast and for expression studies in Chinese Hamster ovary cells. 3. To determine the 3-D structures of E. coil, human and/or mouse NAGS and to investigate the structures of the active site and their catalytic and regulation mechanisms. Crystals of E. coli and mammalian NAGS will be obtained and crystallographic studies will be performed to determine the structure and propose a catalytic mechanism. 4. To develop a knockout mouse for NAGS deficiency and to characterize its phenotype. We will investigate if this mouse could become a conditional hyperammonemia model while on and off carbamylglutamate, a stable functional analog of NAG. 5. To investigate the molecular basis for inherited NAGS deficiency and study genotype/phenotype correlation of the molecular defects. Patients with hyperammonemia will be screened for mutations in the NAGS gene and the mutations found will be characterized and correlated with the clinical and biochemical phenotype, including stable isotope studies for residual ureagenesis capacity.